Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of high mass proteins (> 100,000 Da), directly deposited on polyethylene membranes, is demonstrated. The spectral quality obtained, using standard sample preparation conditions, is equal or superior to that obtained with metal sample stages. Compared to the use of poly(vinylidene difluoride) transfer membranes, this material allows the acquisition of excellent quality MALDI mass spectra from high-mass proteins with a standard UV laser. This gain in capability is not at the expense of either mass accuracy or signal reproducibility; both approach that obtained with standard sample preparations on stainless steel. In addition, for the applications shown, the use of PE as a sample support reduces the severe ion suppression effects typically observed in the MALDI analysis of high-mass mixtures. This permits more precise mass measurements to be made via the use of internal calibration and is illustrated by the mass measurement of a chimeric mouse/human antibody (MW approximately 150,000 Da) by coaddition of bovine albumin dimer (MW approximately 130,000 Da).
The Maillard or browning reaction between reducing sugars and protein contributes to the chemical deterioration and loss of nutritional value of proteins during food processing and storage. This article presents and discusses evidence that the Maillard reaction is also involved in the chemical aging of long-lived proteins in human tissues. While the concentration of the Amadori adduct of glucose to lens protein and skin collagen is relatively constant with age, products of sequential glycation and oxidation of protein, termed glycoxidation products, accumulate in these long-lived proteins with advancing age and at an accelerated rate in diabetes. Among these products are the chemically modified amino acids, N epsilon-(carboxymethyl)lysine (CML), N epsilon-(carboxymethyl)hydroxylysine (CMhL), and the fluorescent crosslink, pentosidine. While these glycoxidation products are present at only trace levels in tissue proteins, there is strong evidence for the presence of other browning products which remain to be characterized. Mechanisms for detoxifying reactive intermediates in the Maillard reaction and catabolism of extensively browned proteins are also discussed, along with recent approaches for therapeutic modulation of advanced stages of the Maillard reaction.
The Maillard or browning reaction between sugar and protein contributes to the increased chemical modification and cross-linking of long-lived tissue proteins in diabetes. To evaluate the role of glycation and oxidation in these reactions, we have studied the effects of oxidative and antioxidative conditions and various types of inhibitors on the reaction of glucose with rat tail tendon collagen in phosphate buffer at physiological pH and temperature. The chemical modifications of collagen that were measured included fructoselysine, the glycoxidation products N epsilon-(carboxymethyl)lysine and pentosidine and fluorescence. Collagen cross-linking was evaluated by analysis of cyanogen bromide peptides using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by changes in collagen solubilization on treatment with pepsin or sodium dodecylsulfate. Although glycation was unaffected, formation of glycoxidation products and cross-linking of collagen were inhibited by antioxidative conditions. The kinetics of formation of glycoxidation products proceeded with a short lag phase and were independent of the amount of Amadori adduct on the protein, suggesting that autoxidative degradation of glucose was a major contributor to glycoxidation and cross-linking reactions. Chelators, sulfhydryl compounds, antioxidants, and aminoguanidine also inhibited formation of glycoxidation products, generation of fluorescence, and cross-linking of collagen without significant effect on the extent of glycation of the protein. We conclude that autoxidation of glucose or Amadori compounds on protein plays a major role in the formation of glycoxidation products and cross-liking of collagen by glucose in vitro and that chelators, sulfhydryl compounds, antioxidants, and aminoguanidine act as uncouplers of glycation from subsequent glycoxidation and cross-linking reactions.
The anti-mitotic taxanes are one of the most important families of anticancer drugs that have broad anti-tumor activity and are widely used for a variety of human cancer types. However, the taxanes are limited by a number of serious pharmacological and toxicological effects, including dose limiting myelosuppression, neurotoxicity and serious hypersensitivity reactions. We are developing a unique delivery system for docetaxel, comprised of a PEGylated liposomal nanoparticle containing a prodrug of docetaxel to improve solubility, tolerability and increase efficacy through improved pharmacokinetics and biodistribution. In this study we evaluated the antitumor activity of MNK-010 on the growth of established human PC3 xenografts in male immunodeficient mice. This study tested the hypothesis that MNK-010 can provide greater efficacy than Taxotere (docetaxel) at equivalent maximum tolerated doses (MTD) in mice implanted with human PC3 tumor cells. Male nude mice bearing PC3 human prostate xenografts were given two or four intravenous (IV) doses of MNK-010, Taxotere or saline. Dosing intervals were twenty-one days for two cycles or every four days for four cycles. The doses of Taxotere and MNK-010 were based on maximum tolerated dose (MTD) or highest dose tested for a given dose interval. Tumor volume was measured twice per week using the Biopticon tumor imaging system and tumor volume data was analyzed to determine tumor growth delay and partial tumor regression. Survival analysis was conducted and median survival time determined. MNK-010 significantly (p <0.05) inhibited PC3 human tumor growth in nude mice at all dose regimens evaluated compared to saline and Taxotere treatment. MNK-010 delayed tumor growth up to 145 days, which was 59 days longer than Taxotere at its MTD. MNK-010 also partially regressed 90% - 100% of tumors in all dose groups. This antitumor activity contributed to achieving median survival of 177 days, which was 150 days longer than saline control and 80 days longer than Taxotere at its MTD. This study demonstrated that Mallinckrodt Pharmaceuticals’ PEGylated liposomal docetaxel prodrug nanoparticle MNK-010 is an active antitumor agent in the PC3 prostate xenograft and possess significantly greater antitumor activity compared to the standard of care Taxotere.
Citation Format: Richard M. Fitch, Jolette K. Wojdyla, James A. Blackledge, William D. McGhee. Anti-tumor activity of liposomal docetaxel prodrug MNK-010 on PC3 human prostate xenografts in mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4400. doi:10.1158/1538-7445.AM2015-4400
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